Apolipoprotein A1 (ApoA1) is together with ApoE an essential component of High Density Lipoprotein particles (HDL), and contributes to formation of the so called ‘good’ cholesterol, as opposed to the Low Density Lipoprotein particles (LDL), often referred to as ‘bad’ cholesterol, composed of ApoB-100, ApoC, ApoE and Apo(A). ApoA1 is a single polypeptide chain consisting of 243 amino acids, synthesised in the liver and the intestine in the form of a pre-pro-protein of 267 amino acid residues (cf. SEQ ID NO:1 (nucleotide sequence) and SEQ ID NO:2 (amino acid sequence) and FIGS. 2a and 2b respectively). The pre-pro-protein is cleaved into a pro-protein that is secreted into the plasma. In the vesicular blood structure, the pro-protein is subsequently processed into its mature (243 AA) form by a calcium-dependent protease (FIG. 2b).
The balance between HDL-cholesterol and LDL-cholesterol in the blood is of the outmost importance to prevent atherosclerosis and related cardiovascular diseases. It has been known for some time that a low level of HDL-cholesterol leads to an increased risk of atherosclerosis, due to the anti-atherosclerosis and anti-hyperlipidemia effect of HDL by stimulating a reverse cholesterol transport (RCT) pathway from the peripheral tissue to the liver.
The sequence of events in RCT is likely as follows (Ohashi et al., QJM. 98(12):845-56, 2005) ApoA-I is produced mainly by the liver, and released into the plasma. Circulating ApoA-I interacts with serum phospholipids and forms nascent discoidal HDL (ndHDL). Once ndHDL is generated, it triggers cholesterol efflux in the macrophages and fibroblasts in the subendothelial space. Externalized cholesterol is absorbed by ndHDL, and subsequently esterified by lecithin:cholesterol acyltransferase (LCAT). HDL particles are enriched with cholesteryl ester and become larger, resulting in HDL3 and HDL2. Phospholipid transfer protein (PLTP) is involved in this process: for example, by fusing two HDL3 into one HDL2 molecule. If HDL molecules are enriched with triglyceride, they are processed by the enzyme hepatic lipase (HL) and become smaller and denser. HL can convert the phospholipid-rich HDL2 to HDL3. However, regulation of the balance of HL and PLTP is not clear. Cholesterol ester transfer protein (CETP) facilitates the equimolar exchange of cholesteryl esters from HDL for triglycerides in apoB100-containing lipoproteins. These cholesteryl esters are then delivered back to the liver via low-density-lipoprotein receptor (LDL-R), converted to bile salts, and eliminated through the gastrointestinal tract.
In addition to this process, when acceptors such as apoA-I and HDL approach macrophages in subintimal space, intracellular cholesterol is released outside the cells for excretion, a process termed cholesterol efflux of macrophages. In this pathway, ATP-binding membrane cassette transport protein A1 (ABCA1) plays a major role in translocating cholesterol into the extracellular space. In addition to ABCA1, four other factors are known to be involved in the pathway. Scavenger receptor B1 (SR-B1) can induce cholesterol efflux by enabling HDL to bind to cells and reorganize lipids within cholesterol-rich domains in the plasma membrane. Caveolins are typically associated with caveolae, which are non-clathrin-coated plasma membrane microdomains rich in cholesterol and glycosphingolipids. Caveolins are small proteins (18-24 kDa) that have a hairpin loop conformation, with both the N and C termini exposed to the cytoplasm. These proteins have the capacity to bind cholesterol, and can transport cholesterol from the endoplasmic reticulum to the plasma membrane. A report showed that over-expression of caveolins enhances cholesterol efflux in hepatic cells without affecting ABCA1 expression, indicating the presence of a caveolin-dependent pathway. Sterol 27-hydroxylase (CYP27A1) is also known as a contributor to cholesterol efflux. CHO cells transfected with CYP27A1 showed increased cholesterol efflux. Since ABCA-1 expression was not altered, CYP27A1 could cause cholesterol efflux independent of other factors. In addition to these pathways, cholesterol efflux can also occur via passive diffusion, in which cholesterol is desorbed down to the concentration gradient onto acceptor molecules. Thus, RCT and cholesterol efflux constitute an efficient pathway by which excess cholesterol can be removed out of the body. Although extensive studies have recently been performed, RCT is a complicated process and its regulation mechanisms are largely unknown. Several key factors described above are involved in the RCT and cholesterol efflux, but the inter-relationship among these factors is not clear.
The role of ApoA1 in this process has been well established over the years and several studies have been directed to the possibilities of administration of recombinant ApoA1 peptide fragments or even full length proteins to subjects with low HDL-cholesterol levels, other dyslipidemic disorders or cardiovascular disease. In order to increase stability of the ApoA1 protein, even thermostabilisation in the presence of chaotropic agents has been tried.
In addition, peptidomimetics of ApoA1 have also been designed that beneficially influence the lipid parameters and/or cholesterol levels in the blood. Several ApoA1 agonists have also been developed in order to mimic the function of ApoA1.
A recombinant ApoA1 mutant protein (the ‘milano’ mutant) is currently under investigation in the treatment of cardiovascular disease (Nissen et al. JAMA 290: 2292-2300, 2003). This mutant ApoA1 has an Arginine to Cysteine mutation at amino acid position 197 (R197C) in the pre-pro-ApoA1 protein amino acid sequence (corresponding to R173C in the mature ApoA1 amino acid sequence). The cysteine in the milano mutant leads to the formation of an ApoA1 dimer, held together by a disulfide bond, due to the additional cysteine residue. It has been established that administration of “nascent” HDL particles reconstructed in vitro by combining phopsholipids and the milano ApoA1 protein to patients leads to a significant decrease in plaque formation. These particles take up the esterified cholesterol from the lipid loaded plaques thereby reducing the size of the atherosclerotic plaques. A similar variant, the ApoA1 ‘paris’ variant has also been identified and has a similar mutation of Arginine to Cysteine, on position 175 (R175C) of the pre-pro-protein (corresponding to R151C in the mature ApoA1 amino acid sequence). The process of making such ApoA1 milano dimers recombinantly or purifying it from plasma samples from carrying humans is however quite cumbersome, in part because of degradation of the protein.
There is therefore clearly a need for a stabilised Apo A1 protein variant that is easy to make recombinantly for use in medicine in the form of a pharmaceutical preparation that improves the inverted cholesterol transport in a patient.